Autoimmunity to stromal-derived autoantigens in rheumatoid ectopic germinal centers exacerbates arthritis and affects clinical response.

Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.

Yishen Tongbi decoction attenuates inflammation and bone destruction in rheumatoid arthritis by regulating JAK/STAT3/SOCS3 pathway.

Background: Yishen-Tongbi Decoction (YSTB), a traditional Chinese prescription, has been used to improve syndromes of rheumatoid arthritis (RA) for many years. Previous research has shown that YSTB has anti-inflammatory and analgesic properties. However, the underlying molecular mechanism of the anti-RA effects of YSTB remains unclear. Purpose and study design: The purpose of this research was to investigate how YSTB affected mice with collagen-induced arthritis (CIA) and RAW264.7 cells induced with lipopolysaccharide (LPS). Results: The findings show that YSTB could significantly improve the clinical arthritic symptoms of CIA mice (mitigate paw swelling, arthritis score, thymus and spleen indices, augment body weight), downregulated expression of pro-inflammatory cytokines like tumor necrosis factor-alpha (TNF-α), interleukin-1ß (IL-1ß), IL-6 and IL-17, while upregulated the level of anti-inflammatory like IL-10 and transforming growth factor-ß (TGF-ß). Meanwhile, YSTB inhibits bone erosion and reduces inflammatory cell infiltration, synovial proliferation, and joint destruction in CIA mice. In addition, we found that YSTB was able to suppress the LPS-induced inflammation of RAW264.7 cells, which was ascribed to the suppression of nitric oxide (NO) production and reactive oxygen species formation (ROS). YSTB also inhibited the production of inducible nitric oxide synthase and reduced the releases of pro-inflammatory cytokines TNF-α, IL-1ß, and IL-6 in LPS-induced RAW264.7 cells. Furthermore, the phosphorylation expression of JAK2, JAK3, STAT3, p38, ERK and p65 protein could be suppressed by YSTB, while the expression of SOCS3 could be activated. Conclusion: Taken together, YSTB possesses anti-inflammatory and prevention bone destruction effects in RA disease by regulating the JAK/STAT3/SOCS3 signaling pathway.

Lingzhi or Reishi Medicinal Mushroom Ganoderma lucidum (Agaricomycetes) Nanogel in a Complete Freund's Adjuvant-Induced Rheumatoid Arthritis Rat Model: Anti-Arthritic, Anti-Inflammatory, and Antioxidative Activity.

Lingzhi or reishi mushroom, Ganoderma lucidum, is a medicinal mushroom quite widely developed as herbal medicine because it has acted as an anticancer, antitumor, antioxidant, and anti-inflammatory. The active mycochemical compounds of G. lucidum mushrooms, such as flavonoids and polysaccharides, can suppress the release of pro-inflammatory cytokines and prevent lipid peroxidation due to oxidative stress. Rheumatoid arthritis (RA) is an autoimmune disease where the exact cause is unknown, and RA prevalence continues to increase yearly. In patients with RA, joint damage and inflammation occur. This study aims to evaluate the effectiveness of G. lucidum nanogels as anti-arthritis, anti-inflammatory, and antioxidative. The research method was a true experiment using a control group and treatment group that randomly assigned, using 24 male Wistar rats (Rattus norvegicus) induced with complete Freund's adjuvant (CFA) 0.1 mL. The rats were divided into six groups; healthy control/HCt (did not receive the treatment), negative control/NCt (induced by CFA), and positive control/PCt (given 0.012 diclofenac sodium). TG1 (given 250 mg G. lucidum nanogels), TG2 (given 500 mg G. lucidum nanogels), TG3 (given 750 mg G. lucidum nanogels). IgG, eNOS, IL-1ß, COX-2, NOS, TNF-α, and IL-6 parameters were measured using ELISA, and the data obtained were analyzed by one-way ANOVA using SPSS (P < 0.05). The results showed that administering G. lucidum nanogels significantly reduced IgG, NOS, TNF-α, COX-2, IL-1ß, and IL-6 and increased eNOS levels. The anti-inflammatory and antioxidative activities in suppressing pro-inflammatory cytokines and increasing eNOS levels prove that the nanogel extract G. lucidum have the potential to be developed as anti-arthritis natural therapeutic.

Fibroblast-like synoviocytes orchestrate daily rhythmic inflammation in arthritis.

Rheumatoid arthritis is a chronic inflammatory disease that shows characteristic diurnal variation in symptom severity, where joint resident fibroblast-like synoviocytes (FLS) act as important mediators of arthritis pathology. We investigate the role of FLS circadian clock function in directing rhythmic joint inflammation in a murine model of inflammatory arthritis. We demonstrate FLS time-of-day-dependent gene expression is attenuated in arthritic joints, except for a subset of disease-modifying genes. The deletion of essential clock gene Bmal1 in FLS reduced susceptibility to collagen-induced arthritis but did not impact symptomatic severity in affected mice. Notably, FLS Bmal1 deletion resulted in loss of diurnal expression of disease-modulating genes across the joint, and elevated production of MMP3, a prognostic marker of joint damage in inflammatory arthritis. This work identifies the FLS circadian clock as an influential driver of daily oscillations in joint inflammation, and a potential regulator of destructive pathology in chronic inflammatory arthritis.

Surface saturation of drug-loaded hollow manganese dioxide nanoparticles with human serum albumin for treating rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease accompanied by energy depletion and accumulation of reactive oxygen species (ROS). Inorganic nanoparticles (NPs) offer great promise for the treatment of RA because they mostly have functions beyond being drug carriers. However, conventional nanomaterials become coated with a protein corona (PC) or lose their cargo prematurely in vivo, reducing their therapeutic efficacy. To avoid these problems, we loaded methotrexate (MTX) into hollow structured manganese dioxide nanoparticles (H-MnO2 NPs), then coated them with a 'pseudo-corona' of human serum albumin (HSA) at physiological concentrations to obtain HSA-MnO2@MTX NPs. Efficacy of MTX, MnO2@MTX, and HSA-MnO2@MTX NPs was compared in vitro and in vivo. Compared to MnO2@MTX, HSA-coated NPs were taken up better by lipopolysaccharide-activated RAW264.7 and were more effective at lowering levels of pro-inflammatory cytokines and preventing ROS accumulation. HSA-MnO2@MTX NPs were also more efficient at blocking the proliferation and migration of fibroblast-like synoviocytes from rats with collagen-induced arthritis. In this rat model, HSA-MnO2@MTX NPs showed better biodistribution than other treatments, specifically targeting the ankle joint. Furthermore, HSA-MnO2@MTX NPs reduced swelling in the paw, regulated pro-inflammatory cytokine production, and limited cartilage degradation and signs of inflammation. These results establish the therapeutic potential of HSA-MnO2@MTX NPs against RA.

[Immunological mechanism of Ermiao Powder improving inflammation in rats with collagen-induced arthritis through α7nAChR-JAK2/STAT3 pathway].

This study investigated the immunological mechanisms of Ermiao powder in the treatment of rheumatoid arthritis rats through the alpha 7 nicotinic acetylcholine receptor(α7nAChR)-Janus kinases 2(JAK2)/signal transducer and activator of transcription 3(STAT3) signaling pathway. A total of 56 female Wistar rats were randomly divided into the normal group(HG, n=8), collagen-induced arthritis(CIA) model group(CM, n=8), vagotomy group(VA, n=8), sham group(SH, n=8), Ermiao Powder treatment model group(EM, n=8), Ermiao Powder treatment for vagotomy group(EV, n=8) and Ermiao Powder treatment for sham group(ES, n=8). Following the establishment of CIA models in all groups except the HG group, the rats underwent unilateral vagotomy and sham operation(only the vagus nerve was separated). Drug treatment was started 7 days after surgery and continued for 35 days. The body weight and joints of rats were recorded, the pathological changes of the spleen of rats were observed, the contents of interleukin-6(IL-6), interleukin-1ß(IL-1ß) and tumor necrosis factor-α(TNF-α) in serum were detected by enzyme-linked immunosorbent assay(ELISA), and the mRNA and protein expression of α7nAChR-JAK2/STAT3 pathway core genes in spleen were detected by qRT-PCR, Western blot and immunohistochemistry. RESULTS:: showed that CM group(compared with HG group) and VA group(compared with CM group and SH group) had significantly decreased body weight(P<0.05, P<0.01), increased arthritis score(P<0.05, P<0.01), swollen ankle joints with deformity, and increased and enlarged lymph nodes in the spleen. There were also notable increases in the serum levels of IL-6, IL-1ß and TNF-α(P<0.05, P<0.01), and in the mRNA expressions of JAK2 and STAT3 in the spleen(P<0.05, P<0.01). The protein levels of phosphorylated JAK2(p-JAK2)/JAK2 and phospho-STAT3(p-STAT3)/STAT3 were significantly increased(P<0.05, P<0.01), and the number of JAK2, p-JAK2, STAT3 and p-STAT3 cells increased(P<0.05, P<0.01). EM group(compared with CM group) and ES group(compared with SH group) exhibited significantly increased body weight(P<0.01), decreased arthritis scores(P<0.05, P<0.01), reduced swelling of ankle joint, and decreased number and volume of lymph nodes in the spleen. Furthermore, serum levels of IL-6, IL-1ß, and TNF-α decreased(P<0.05, P<0.01), the mRNA expression of JAK2 and STAT3 in spleen decreased(P<0.05, P<0.01), the protein levels of p-JAK2/JAK2 and p-STAT3/STAT3 decreased(P<0.05, P<0.01), and the number of JAK2, p-JAK2, STAT3 and p-STAT3 cells decreased(P<0.05, P<0.01), whereas the mRNA and protein expressions of α7nAChR were significantly increased(P<0.01). Compared with the VA group, there was no significant differences in weight gain and arthritis scores in the EV group. The number of lymph nodes in the spleen was not significantly reduced and the volume was still large, suggesting the inflammation was not significantly improved. The serum levels of IL-6, IL-1ß and TNF-α were not significantly different, and there were no significant differences in α7nAChR, JAK2, and STAT3 mRNA expression in the spleen. The protein expression levels of p-JAK2/JAK2 and α7nAChR in spleen were lower(P<0.05, P<0.01), while p-STAT3/STAT3 protein expression was not significantly different. Besides, the two groups had no significant difference in the number of JAK2, p-JAK2, STAT3, and p-STAT3 cells. The results suggested that unilateral vagotomy promoted the increase of phosphorylated JAK2 and STAT3 expressions and exacerbated inflammation. In contrast, Ermiao Powder alleviated the inflammation in rheumatoid arthritis rats by activating the α7nAChR-mediated JAK2/STAT3 pathway through the vagus nerve, suggesting that the α7nAchR-JAK2/STAT3 pathway may be a potential target for the treatment of rheumatoid arthritis.

[Effect of triptolide on reproductive toxicity in female rats with â ¡ type collagen induced arthritis].

In order to study the toxic effect and mechanism of triptolide(TP) on the reproductive system of female rats with Ⅱ type collagen induced arthritis(CIA), 50 SD rats were randomly divided into normal control group, CIA model group, and three groups receiving TP tablets at clinically equivalent doses of 0. 5, 1, and 2 times, respectively(with TP dosages of 3. 75, 7. 5, and 15 µg·kg~(-1)·d~(-1)), each comprising 10 rats. Intragastric administration was started on the day after the first immunization, once a day, for 42 days.The results were taken on the 21st and 42nd days to calculate the uterine and ovarian organ indexes; pathological and morphological changes in uterus and ovaries were observed under a light microscope; and the levels of estradiol(E_2) and cytochrome P450A1(aromatase,CYP19A1) in ovarian homogenate were detected by ELISA. Furthermore, immunohistochemistry was employed to detect the expression levels of transforming growth factor ß3( TGFß3) pathway-related proteins, mothers against decapentaplegic homolog 3(Smad3) and steroidogenic factor-1(SF-1) in ovarian tissues. In vitro, the mouse Chinese hamster ovary(CHO) cell line was established, and after 24 hours of TP administration(30, 60, 120 nmol·L~(-1)), cell proliferation was detected by the thiazolyl blue tetrazolium bromide(MTT) method, apoptosis by the flow cytometry, and TGFß3, Smad3 and SF-1 protein expression in cells by the Western blot method, and the nuclear entry of SF-1 was detected by immunofluorescence. The results showed that compared with the CIA model group, all TP administration groups showed decreased number of uterine glands, total follicles, mature follicles, and corpus luteum on days 21 and 42 of administration, but there was no statistical difference, and only the administration of 2 times the clinically equivalent dose of TP could significantly increase the number of atretic follicles at 42 days of administration. TP at 3. 75 µg·kg-1·d-1significantly reduced the level of E_2 at 21 days of administration and the expression of TGFß3 and Smad3 factors in ovarian tissues,but had no significant effect on the rate-limiting enzyme in estrogen synthesis CYP19A1. TP at 7. 5 and 15 µg·kg~(-1)·d~(-1) significantly reduced the expression of SF-1 regardless of administration for 21 days or 42 days. TP can significantly promote ovarian cell apoptosis in vitro, with apoptosis mainly concentrated in the late stage of apoptosis after 24 hours of administration. In addition, 60 nmol·L~(-1) TP significantly reduced the protein expression of TGFß3, Smad3 and SF-1 in a dose-dependent manner. In summary, intragastric administration of TP at less than 2 times the clinically equivalent dose for 21 days and 42 days did not cause obvious reproductive damage to the uterus and ovarian tissues of CIA rats, and the number of atretic follicles changed significantly only when the 2 times the clinically equivalent dose was administered for 42 days. TP exerted reproductive toxicity in vivo on reproductive target organs and in vitro on ovarian cells by inhibiting the expression of TGFß3/Smad3/SF-1 pathway.

Selection and Mechanism Study of Q-Markers for Xanthocerais lignum Anti-Rheumatoid Arthritis Based on Serum Spectrum-Effect Correlation Analysis.

OBJECTIVE: To elucidate the chemical profile of Xanthocerais lignum's extracts of different polarities and their impact on rheumatoid arthritis (RA), we identified anti-RA markers and predicted their action mechanisms. METHODS: A collagen-induced arthritis rat model was established, and UPLC-Q-Exactive Orbitrap MS technology was employed to analyze and identify the chemical constituents within the alcohol extract of Xanthocerais lignum and its various extraction fractions, as well as their translocation into the bloodstream. Serum spectrum-effect correlation analysis was utilized to elucidate the pharmacodynamic material basis of Xanthocerais lignum against RA and to screen for Q-Markers. Finally, the potential anti-RA mechanisms of the Q-Markers were predicted through compound-target interaction data and validated using molecular docking techniques. RESULTS: We identified 71 compounds, with flavan-3-ols and flavanones as key components. Of these, 36 were detected in the bloodstream, including 17 original and 19 metabolized forms. Proanthocyanidin A2, dihydroquercetin, catechin, and epicatechin (plus glucuronides) showed potential anti-RA activity. These compounds, acting as Q-Markers, may modulate ERK, NF-κB, HIF-1α, and VEGF in the HIF-1 pathway. CONCLUSIONS: This research clarifies Xanthocerais lignum's pharmacodynamic material basis against RA, identifies 4 Q-Markers, and offers insights into their mechanisms, aiding quality assessment and lead compound development for RA treatment.

[Identification of potential biomarkers and immunoregulatory mechanisms of rheumatoid arthritis based on multichip co-analysis of GEO database].

OBJECTIVE: To identify the biomarkers for early rheumatoid arthritis (RA) diagnosis and explore the possible immune regulatory mechanisms. METHODS: The differentially expressed genesin RA were screened and functionally annotated using the limma, RRA, batch correction, and clusterProfiler. The protein-protein interaction network was retrieved from the STRING database, and Cytoscape 3.8.0 and GeneMANIA were used to select the key genes and predicting their interaction mechanisms. ROC curves was used to validate the accuracy of diagnostic models based on the key genes. The disease-specific immune cells were selected via machine learning, and their correlation with the key genes were analyzed using Corrplot package. Biological functions of the key genes were explored using GSEA method. The expression of STAT1 was investigated in the synovial tissue of rats with collagen-induced arthritis (CIA). RESULTS: We identified 9 core key genes in RA (CD3G, CD8A, SYK, LCK, IL2RG, STAT1, CCR5, ITGB2, and ITGAL), which regulate synovial inflammation primarily through cytokines-related pathways. ROC curve analysis showed a high predictive accuracy of the 9 core genes, among which STAT1 had the highest AUC (0.909). Correlation analysis revealed strong correlations of CD3G, ITGAL, LCK, CD8A, and STAT1 with disease-specific immune cells, and STAT1 showed the strongest correlation with M1-type macrophages (R=0.68, P=2.9e-08). The synovial tissues of the ankle joints of CIA rats showed high expressions of STAT1 and p-STAT1 with significant differential expression of STAT1 between the nucleus and the cytoplasm of the synovial fibroblasts. The protein expressions of p-STAT1 and STAT1 in the cell nuclei were significantly reduced after treatment. CONCLUSION: CD3G, CD8A, SYK, LCK, IL2RG, STAT1, CCR5, ITGB2, and ITGAL may serve as biomarkers for early diagnosis of RA. Gene-immune cell pathways such as CD3G/CD8A/LCK-γδ T cells, ITGAL-Tfh cells, and STAT1-M1-type macrophages may be closely related with the development of RA.

Impact of Interleukin-6 Activation and Arthritis on Epidermal Growth Factor Receptor (EGFR) Activation in Sensory Neurons and the Spinal Cord.

In tumor cells, interleukin-6 (IL-6) signaling can lead to activation of the epidermal growth factor receptor (EGFR), which prolongs Stat3 activation. In the present experiments, we tested the hypothesis that IL-6 signaling activates EGFR signaling in peripheral and spinal nociception and examined whether EGFR localization and activation coincide with pain-related behaviors in arthritis. In vivo in anesthetized rats, spinal application of the EGFR receptor blocker gefitinib reduced the responses of spinal cord neurons to noxious joint stimulation, but only after spinal pretreatment with IL-6 and soluble IL-6 receptor. Using Western blots, we found that IL-6-induced Stat3 activation was reduced by gefitinib in microglial cells of the BV2 cell line, but not in cultured DRG neurons. Immunohistochemistry showed EGFR localization in most DRG neurons from normal rats, but significant downregulation in the acute and most painful arthritis phase. In the spinal cord of mice, EGFR was highly activated mainly in the chronic phase of inflammation, with localization in neurons. These data suggest that spinal IL-6 signaling may activate spinal EGFR signaling. Downregulation of EGFR in DRG neurons in acute arthritis may limit nociception, but pronounced delayed activation of EGFR in the spinal cord may be involved in chronic inflammatory pain.

Exploring the supplementary potential of all-trans retinoic acid with methotrexate in rheumatoid arthritis: modulation of synovial cell apoptosis and autophagy.

OBJECTIVES: The imbalance between apoptosis and proliferation in fibroblast-like synoviocytes (FLSs) plays a key role in the pathogenesis of rheumatoid arthritis (RA). This study aims to investigate the potential of all-trans retinoic acid (ATRA) as a supplementary therapeutic agent alongside methotrexate (MTX) for RA, by examining its ability to inhibit synovial cell proliferation and enhance apoptosis through the ROS-JNK signalling pathway. METHODS: The viability, apoptosis, and autophagy levels of human rheumatoid arthritis fibroblast-like synovial cells (HFLS-RA) were evaluated, while ROS generation was measured through the DCFH-DA fluorescence microplate assay. Western blotting was used to analyse the expression levels of JNK signalling pathway-related proteins. To assess therapeutic potential in vivo, a collagen-induced arthritis (CIA) model was established in Wistar rats. RESULTS: Small doses of MTX did not significantly affect the viability of HFLS-RAs or induce apoptosis. However, when ATRA was added to the treatment, the therapy markedly inhibited cell proliferation and induced apoptosis and excessive autophagy. Mechanistically, ATRA activated the ROS/JNK signalling pathway in HFLS-RAs. ROS scavengers and JNK inhibitors significantly attenuated ATRA-induced apoptosis and autophagy. In vivo, the combination therapy demonstrated a remarkable enhancement of the anti-arthritic efficacy in CIA rats. CONCLUSIONS: The ability of ATRA to inhibit proliferation in RA FLSs through autophagy and apoptosis underscores its potential as a supplementary therapeutic agent alongside MTX for RA, particularly when compared to the limited impact of MTX on these processes. This combined strategy holds promise for enhancing therapeutic outcomes and warrants further investigation in the management of RA.

[Terminalia chebula water extract reduces joint inflammation by regulating cellular immunity in spleen cells of collagen-induced arthritis (CIA) rats through up-regulation of PD-1/PD-L1].

Objective To investigate the effect of Terminalia chebula water extract (TCWE) on the cellular immunity and PD-1/PD-L1 pathway in rats with collagen-induced arthritis (CIA). Methods SD rats were randomly divided into four groups: a control group, a CIA group, a TCWE group and a methotrexate (MTX) group, with 15 rats in each group. Except for the control group, SD rats in other groups were subcutaneously injected with type II collagen to establish the model of collagen-induced arthritis (CIA). The rats in the TCWE group were treated with 20 mg/(kg.d) TCWE and the rats in the MTX group were treated with 1.67 mg/(kg.d) MTX. After 14 days of treatment, the cartilage morphology was examined using hematoxylin-eosin (HE) staining, and splenic T lymphocyte apoptosis and Treg/Th17 cell ratio were detected by flow cytometry. The mRNA expressions of retinoid-related orphan nuclear receptor γt (RORγt), forkhead box P3 (FOXP3), PD-1 and PD-L1 in spleen were detected by reverse transcription PCR. The expression and localization of RORγt and FOXP3 were detected by immunohistochemical staining. The protein expressions of PD-1 and PD-L1 in splenic lymphocytes were detected by Western blot, and the levels of serum interleukin 17 (IL-17) and transforming growth factor ß (TGF-ß) in rats were detected by ELISA. Results Compared with CIA group, the pathological changes of cartilage and synovium were significantly alleviated in the TCWE group and the MTX group. Both the apoptosis rate of T lymphocytes in spleen and the ratio of Treg/Th17 cells increased. The expression of RORγt decreased, while the expressions of FOXP3, PD-1 and PD-L1 increased in spleen lymphocytes. The level of serum IL-17 decreased, while the level of serum TGF-ß increased. Conclusion TCWE treatment may activate PD-1/PD-L1 pathway in spleen cells to regulate cellular immunity, thus reducing cartilage injury in CIA rats.

Adipose-derived stem cell exosomes loaded with icariin alleviates rheumatoid arthritis by modulating macrophage polarization in rats.

Rheumatoid arthritis (RA) is a chronic autoimmune disease marked by synovitis and cartilage destruction. The active compound, icariin (ICA), derived from the herb Epimedium, exhibits potent anti-inflammatory properties. However, its clinical utility is limited by its water insolubility, poor permeability, and low bioavailability. To address these challenges, we developed a multifunctional drug delivery system-adipose-derived stem cells-exosomes (ADSCs-EXO)-ICA to target active macrophages in synovial tissue and modulate macrophage polarization from M1 to M2. High-performance liquid chromatography analysis confirmed a 92.4 ± 0.008% loading efficiency for ADSCs-EXO-ICA. In vitro studies utilizing cellular immunofluorescence (IF) and flow cytometry demonstrated significant inhibition of M1 macrophage proliferation by ADSCs-EXO-ICA. Enzyme-linked immunosorbent assay, cellular transcriptomics, and real-time quantitative PCR indicated that ADSCs-EXO-ICA promotes an M1-to-M2 phenotypic transition by reducing glycolysis through the inhibition of the ERK/HIF-1α/GLUT1 pathway. In vivo, ADSCs-EXO-ICA effectively accumulated in the joints. Pharmacodynamic assessments revealed that ADSCs-EXO-ICA decreased cytokine levels and mitigated arthritis symptoms in collagen-induced arthritis (CIA) rats. Histological analysis and micro computed tomography confirmed that ADSCs-EXO-ICA markedly ameliorated synovitis and preserved cartilage. Further in vivo studies indicated that ADSCs-EXO-ICA suppresses arthritis by promoting an M1-to-M2 switch and suppressing glycolysis. Western blotting supported the therapeutic efficacy of ADSCs-EXO-ICA in RA, confirming its role in modulating macrophage function through energy metabolism regulation. Thus, this study not only introduces a drug delivery system that significantly enhances the anti-RA efficacy of ADSCs-EXO-ICA but also elucidates its mechanism of action in macrophage function inhibition.

Comparison of disease-modifying anti-rheumatic drugs and hyperbaric oxygen therapy in the experimental model of rheumatoid arthritis in rats.

In this study, we wanted to investigate the effectiveness of combining disease-modifying anti-rheumatic drugs (DMARD) with hyperbaric oxygen therapy (HBOT) in reducing inflammation in a rheumatoid arthritis (RA) model using rats. We divided 56 male Sprague-Dawley rats into seven groups and induced RA using complete Freund's adjuvant. Some groups received HBOT, whereas others were given etanercept or leflunomide. We started the treatment on the 10th day after inducing RA and continued it for 18 days. To evaluate the effectiveness of the treatments, we measured paw swelling and used X-rays to examine the joints before and after the treatment. We also analysed the levels of two inflammatory markers, tumour necrosis factor (TNF)-α and interleukin (IL)-1ß, using an enzyme-linked immunosorbent assay. Additionally, we conducted histological analysis and assessed the expressions of anti-IL-1ß and anti-TNF-α antibodies. All the treatment groups showed a significant decrease in arthritis scores, paw swelling and levels of TNF-α and IL-1ß. The X-ray images revealed improvements in joint structure, and the histopathological analysis showed reduced inflammation and collagen abnormalities. Combining DMARD with HBOT had similar effects to individual therapies, suggesting a cost-effective and potentially safer approach for improving outcomes in rats with RA.

Graphene oxide quantum dots-loaded sinomenine hydrochloride nanocomplexes for effective treatment of rheumatoid arthritis via inducing macrophage repolarization and arresting abnormal proliferation of fibroblast-like synoviocytes.

The characteristic features of the rheumatoid arthritis (RA) microenvironment are synovial inflammation and hyperplasia. Therefore, there is a growing interest in developing a suitable therapeutic strategy for RA that targets the synovial macrophages and fibroblast-like synoviocytes (FLSs). In this study, we used graphene oxide quantum dots (GOQDs) for loading anti-arthritic sinomenine hydrochloride (SIN). By combining with hyaluronic acid (HA)-inserted hybrid membrane (RFM), we successfully constructed a new nanodrug system named HA@RFM@GP@SIN NPs for target therapy of inflammatory articular lesions. Mechanistic studies showed that this nanomedicine system was effective against RA by facilitating the transition of M1 to M2 macrophages and inhibiting the abnormal proliferation of FLSs in vitro. In vivo therapeutic potential investigation demonstrated its effects on macrophage polarization and synovial hyperplasia, ultimately preventing cartilage destruction and bone erosion in the preclinical models of adjuvant-induced arthritis and collagen-induced arthritis in rats. Metabolomics indicated that the anti-arthritic effects of HA@RFM@GP@SIN NPs were mainly associated with the regulation of steroid hormone biosynthesis, ovarian steroidogenesis, tryptophan metabolism, and tyrosine metabolism. More notably, transcriptomic analyses revealed that HA@RFM@GP@SIN NPs suppressed the cell cycle pathway while inducing the cell apoptosis pathway. Furthermore, protein validation revealed that HA@RFM@GP@SIN NPs disrupted the excessive growth of RAFLS by interfering with the PI3K/Akt/SGK/FoxO signaling cascade, resulting in a decline in cyclin B1 expression and the arrest of the G2 phase. Additionally, considering the favorable biocompatibility and biosafety, these multifunctional nanoparticles offer a promising therapeutic approach for patients with RA.

Undenatured type II collagen protects against collagen-induced arthritis by restoring gut-joint homeostasis and immunity.

Oral administration of harmless antigens can induce suppression of reactive immune responses, a process that capitalises on the ability of the gastrointestinal tract to tolerate exposure to food and commensal microbiome without triggering inflammatory responses. Repeating exposure to type II collagen induces oral tolerance and inhibits induction of arthritis, a chronic inflammatory joint condition. Although some mechanisms underlying oral tolerance are described, how dysregulation of gut immune networks impacts on inflammation of distant tissues like the joints is unclear. We used undenatured type II collagen in a prophylactic regime -7.33 mg/kg three times/week- to describe the mechanisms associated with protective oral immune-therapy (OIT) in gut and joint during experimental Collagen-Induced Arthritis (CIA). OIT reduced disease incidence to 50%, with reduced expression of IL-17 and IL-22 in the joints of asymptomatic mice. Moreover, whilst the gut tissue of arthritic mice shows substantial damage and activation of tissue-specific immune networks, oral administration of undenatured type II collagen protects against gut pathology in all mice, symptomatic and asymptomatic, rewiring IL-17/IL-22 networks. Furthermore, gut fucosylation and microbiome composition were also modulated. These results corroborate the relevance of the gut-joint axis in arthritis, showing novel regulatory mechanisms linked to therapeutic OIT in joint disease.

Anti-Inflammatory and Immunomodulatory Effects of 0.1 Sub-Terahertz Irradiation in Collagen-Induced Arthritis Mice.

The primary emphasis of photoimmunology is the impact of nonionizing radiation on the immune system. With the development of terahertz (THz) and sub-terahertz (sub-THz) technology, the biological effects of this emerging nonionizing radiation, particularly its influence on immune function, remain insufficiently explored but are progressively attracting attention. Here, we demonstrated that 0.1 sub-THz radiation can modulate the immune system and alleviate symptoms of arthritis in collagen-induced arthritis (CIA) mice through a nonthermal manner. The application of 0.1 sub-THz irradiation led to a decrease in proinflammatory factors within the joints and serum, reducing the levels of blood immune cells and the quantity of splenic CD4+ T cells. Notably, 0.1 sub-THz irradiation restored depleted Treg cells in CIA mice and re-established the Th17/Treg equilibrium. These findings suggested that sub-THz irradiation plays a crucial role in systemic immunoregulation. Further exploration of its immune modulation mechanisms revealed the anti-inflammatory properties of 0.1 sub-THz on LPS-stimulated skin keratinocytes. Through the reduction in NF-κB signaling and NLRP3 inflammasome activation, 0.1 sub-THz irradiation effectively decreased the production of inflammatory factors and immune-active substances, including IL-1ß and PGE2, in HaCaT cells. Consequently, 0.1 sub-THz irradiation mitigated the inflammatory response and contributed to the maintenance of immune tolerance in CIA mice. This research provided significant new evidence supporting the systemic impacts of 0.1 sub-THz radiation, particularly on the immune system. It also enhanced the field of photoimmunology and offered valuable insights into the potential biomedical applications of 0.1 sub-THz radiation for treating autoimmune diseases.

Withaferin A alleviates inflammation in animal models of arthritis by inhibiting the NF-κB pathway and cytokine release.

Withaferin A, a steroid lactone from Withania somnifera, exhibits anti-inflammatory, immunomodulatory, and antioxidant properties. This study investigated the effects of withaferin A on collagen-induced arthritis (CIA) rats, focusing on NF-κB p65 regulation and cytokine release. Withaferin A (50 mg/kg b.wt., orally) or methotrexate (0.25 mg/kg b.wt., i.p., as a reference drug) was given to CIA rats daily for 20 days postarthritis induction. Joints were removed from nonarthritic and arthritic rats to assess the levels of NO, MPO, interleukin (IL)-1ß, IL-6, IL-10, TNF-α, COX-2, and NF-κB via ELISA. Furthermore, the mRNA expression of IL-1ß, IL-10, TNF-α, COX-2, iNOS, and NF-κB was also assessed through qPCR. Treatment with withaferin A significantly inhibited the levels of inflammatory cytokines and the transcription factor NF-κB; suppressed the expression of IL-1ß, IL-10, TNF-α, COX-2, iNOS, and NF-κB in the joint tissue of CIA rats; and reduced cartilage and bone destruction, as shown by H&E staining. To confirm the results obtained from biochemical and molecular studies and to determine the molecular target of withaferin A, we performed a molecular simulation of the potential targets of withaferin A, which identified the NF-κB pathway as its target. These results suggested that withaferin A effectively attenuated rheumatoid arthritis progression by inhibiting the activation of the NF-κB pathway and the downstream secretion of inflammatory cytokines.

Alginate nanogel-embedded liposomal drug carriers facilitate drug delivery efficiency in arthritis treatment.

Despite numerous advantages of liposomes in treating rheumatoid arthritis (RA), the in vivo stability remains a critical issue. Current strategies for improving liposomal stability often compromise their original properties. Herein, we designed an alginate nanogel-embedded liposome aiming at retaining those inherent advantages while enhancing their in vivo stability. The introduction of alginate network within the liposome core can provide mechanical support and controlled drug release without affecting the surface properties. Results showed the cross-linking of alginate network within the inner core of liposomes elevated the particle rigidity to 3 times, allowing for improved stability and decreased drug leakage. Moreover, this nanogel-embedded liposome with optimized elasticity obviously facilitated cellular uptake in inflammatory macrophages. When entering blood circulation, increased rigidity altered the composition of protein corona on the particle surface, resulting in 2-fold increase in circulation time and improved drug accumulation in arthritic joints. When anti-inflammatory chlorogenic acid (CA) was encapsulated into the nanogel network, this CA-loaded nanogel-embedded liposome significantly inhibited ROS production and inflammatory response, ultimately achieved superior therapeutic outcome in arthritic rats. Results demonstrated that this nanogel-embedded liposomes can essentially retain the inherent advantages and overcome the drawbacks of liposomes, thereby improving the drug delivery efficiency.

Sodium chloride promotes macrophage pyroptosis and aggravates rheumatoid arthritis by activating SGK1 through GABA receptors Slc6a12.

Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial inflammation and the production of autoantibodies. Previous studies have indicated an association between high-salt diets (HSD) and an increased risk of RA, yet the underlying mechanisms remain unclear. Macrophage pyroptosis, a pro-inflammatory form of cell death, plays a pivotal role in RA. In this study, we demonstrate that HSD exacerbates the severity of arthritis in collagen-induced arthritis (CIA) mice, correlating with macrophage infiltration and inflammatory lesions. Given the significant alterations observed in macrophages from CIA mice subjected to HSD, we specifically investigate the impact of HSD on macrophage responses in the inflammatory milieu of RA. In our in vitro experiments, pretreatment with NaCl enhances LPS-induced pyroptosis in RAW.264.7 and THP-1 cells through the p38 MAPK/NF-κB signaling pathway. Subsequent experiments reveal that Slc6a12 inhibitors and SGK1 silencing inhibit sodium-induced activation of macrophage pyroptosis and the p38 MAPK/NF-κB signaling pathway, whereas overexpression of the SGK1 gene counteracts the effect of sodium on macrophages. In conclusion, our findings verified that high salt intake promotes the progression of RA and provided a detailed elucidation of the activation of macrophage pyroptosis induced by sodium transportation through the Slc6a12 channel.